Biochemical and mechanical stimuli for improved material properties and preservation of tissue-engineered cartilage

The focus of this research and overall hypothesis is that bioreactors that employ both perfusion and shear will improve chondrogenesis and preservation to produce functionally relevant cartilage by modulating shear stress and introducing exogenous preservation factors. Applying both a low shear stress across the surface of cell-seeded scaffolds and perfusion through them in a perfusion concentric cylinder (PCC) bioreactor may stimulate chondrocytes to undergo chondrogenesis. Experimental data showed that the PCC bioreactor stimulated cartilage growth over the course of four weeks, supported by the appearance of glycosaminoglycan (GAG) and collagen type II, which are markers for articular cartilage. Computational fluid dynamics modeling showed that shear stress across the face of the construct was heterogeneous, and that only the center experienced a relatively uniform shear stress of 0.4 dynes/cm 2 when the outer cup of the bioreactor rotated at 38 rpm. When compared to a concentric cylinder (CC) bioreactor that employed only shear stress, the PCC bioreactor caused a significant increase in cellular proliferation, which resulted in a 12-fold increase in cell number per construct compared to 7-fold increase within the CC bioreactor. However, the PCC bioreactor had a less pronounced effect on glycosaminoglycan and collagen content with 1.3 mg of GAG and 1.8 mg of collagen per construct within the CC bioreactor and 0.7 mg of GAG and 0.8 mg of collagen per construct within the PCC bioreactor after 28 days in culture (p < 0.05). Our results led to an important observation that the PCC bioreactor affected cellular proliferation significantly but not extracellular matrix synthesis.;The next objective of this study focused on the PCC bioreactor to evaluate the direct role of perfusion and shear on chondrogenesis in vitro and in vivo. In the presence of perfusion+shear, cellular growth increased by 33 fold, while compared to the presence of shear alone, cellular growth increased by 11 fold (p < 0.05). GAG deposition per construct was not affected by either bioreactors, both producing 1.2 mg/construct by Day 28. Total collagen deposition, however, was significantly higher in the perfusion+shear bioreactor with 2.8 mg/construct, while the shear bioreactor produced 1.2 mg/construct by Day 28. The compressive and shear modulus measured showed no difference between the two bioreactors and were approximately 0.15 MPa and 0.33 MPa, respectively. However, the perfusion+shear bioreactor had a significantly higher phase angle compared to the shear bioreactor, due to greater collagen deposition per construct in the perfusion+shear bioreactor. This observation combined with the PCC versus CC study suggests that directional shear stress may influence the proliferation rate of chondrocytes, while the magnitude of shear stress may influence total collagen deposition.;A rat xiphoid chondral defect model was developed to assess in vivo chondrogenesis supported by tissue-engineered cartilage constructs (TECCs) grown under perfusion+shear conditions, shear only, or static conditions for 28 days. Implanted into the defect, the tissue-engineered cartilage cultured in the mechanically active bioreactors resulted in more mature engineered cartilage over static cultured constructs after 28 days in vivo. This observation showed the necessity to consider mechanical forces and cell density when culturing engineered cartilage for implantation into cartilage defects.;A major challenge for cartilage tissue engineering is the development of viable preservation methods to ensure long-term "off-the-shelf" availability. The objective of the final study was to identify variables in ice-free cryopreservation by vitrification that affect cell viability of tissue-engineered cartilage and determine whether preservation could be performed in a bioreactor. The final results showed that VS70 preserved 2.2 times more viable cells than VS55 and VS55 preserved 2.8 times more viable cells than VS83. The reduction of steps from VS70 in 6/7 sequential steps to VS70 in 4/4 steps did not alter cell viability, neither did the incorporation of the PCC bioreactor to better permeate VS70 into TECCs. The highest cell viabilities reached were 47.8 +/- 5.6% in the PCC bioreactor treatment, which demonstrated the need to further tailor vitrification protocols to increase cell viability and TECCs function. (Abstract shortened by UMI.)...